Process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and of anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form

ABSTRACT

The invention relates to a process for producing and obtaining anaphylatoxin- and cocytotaxin-containing leucotaxine preparations and anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form from contact-activated mammalian serum by the following steps: 
     separation of the proteins from other serum constituents to obtain a serum protein concentrate fraction, 
     separation of a part of accompanying foreign blood proteins from anaphylatoxin and cocytotaxin present in the said protein concentrate fraction, 
     isolation of the leucotaxine preparation by chromatography on hydroxyapatite, 
     and optionally further purification and/or separation of the leucotaxine preparation into anaphylatoxin and cocytotaxin proteins by chromatographical methods. 
     The invention is characterized in that, prior to chromatography on hydroxyapatite, in the above-mentioned process, a major fraction of accompanying foreign blood constituents is separated from the said serum protein concentrate fraction by fractional elution and/or precipitation with a water-soluble alcohol and/or at least one molecular sieve filtration.

This is a continuation of application Ser. No. 275,014, filed June 18,1981, now U.S. Pat. No. 4,495,096.

BACKGROUND OF THE INVENTION

The present invention relates to a method of separating bloodcomponents. More specifically it relates to a method of producing andobtaining anaphylatoxin- and cocytotaxin-containing leucotaxinepreparations and of anaphylatoxin and cocytotaxin proteins inmolecularly homogeneous, biologically active form.

Destruction of tissue in inflammation induced by non-imunological and/orimmunological processes leads to formation of a variety of endogenoussubstances (mediators and hormones). They regulate the complexindividual steps of activation of inflammation and tissue repairprocesses. The mediators are produced either as humoral mediators bylimited and regulated proteolysis of plasma or serum proteins, or theyare released as cellular mediators by active secretion and/or cell lysisfrom cells and tissues. They form part of the body's defensive system,which systemic and local activation they play part.

They thus contribute to removal and detoxification of destroyedendogenous substances and/or invaded foreign bodies. In addition, byregulation of the cell-division and tissue growth processes in woundhealing, they participate in restoration of the physiological structuresand functions of the organism.

Like the classical hormones of the endocrinal glands, inflammatorymediators are trace substances, that are present in situ in only minuteconcentrations in tissue or blood.

By activation of the kinin system, the coagulation system, thecomplement system, and also of other blood-protein and cell factors, avariety of mediators may be produced concurrently or sequentially whichare responsible for the apparent biological activities of activatedserum. Amongst them to mention are chemical attraction of leucocytes(leucotaxis), immunoadherence and the smooth muscle concentration.Anaphylatoxin and cocytotaxin are among the blood protein mediatorsformed by limited, regulated proteolysis of plasma and serum factors inconcurrence with complement system activation. They play a major role inthe chemical attraction of leucocytes. In addition, anaphylatoxin alsopossesses pharmacological properties and cardiovascular effects onaccount of its spasmogenic effects on muscle cells.

Anaphylatoxin was descovered after treating mammalian sera withantigen-antibody complexes; cf. E. Friedberger, Z. Immunonitatsforsch. 4(1919), p. 636-689. It is considered as one of the fragments (C5a) ofthe fifth complement component, with which it has many biologicalactivities in common; cf. J. Jensen, Science 155 (1967), p. 1122-1123.There is still no proof of their chemical identity. Formation ofanaphylatoxin and other mediators can be induced by contact reactions ofmammalian blood, plasma, or serum with various hydrophilic, insolublehigh-molecular substances, such as dextran, yeast, and also withbacterial endotoxins (lipopolysaccharides). Application of such modifiedsera in vivo and in vitro induces various types of biological effects.Amongst them to mention are the typical anaphylatoxin effects and otherreactions which are similar to, or comparable with in vivo immune andnon-immune processes apparent in allergy and tissue damage reactions.These biological reactions in particular include release of histamine,lethal shock, contraction of the smooth muscles, and chemotacticactivity for neutrophil and eosinophil leucocytes.

A process of ten steps for obtaining anaphylatoxin from rat, pig andguinea-pig serum treated with dextran, yeast or immune complexes, hasbeen described by J. H. Wissler in Eur. J. Immunol. 2 (1972), pp. 73-96and in Int. Arch. Allergy 32 (1972), pp. 722-747. According to thisknown process, anaphylatoxin is separated from accompanying foreignproteins by chromatography on hydroxyapatite. Prior to this step, ananaphylatoxin containing crude protein fraction of contact-activatedserum is separated off by cation exchange reaction from the supernatant,negatively absorbed serum components. The anaphylatoxin-containingprotein eluate obtained by the cation exchange reaction is concentratedby salting-out precipitation of proteins with ammonium sulfate (serumprotein concentrate fraction). Prior to chromatography on hydroxyapatitepart of the foreign substances, including a small fraction ofaccompanying foreign proteins, are removed by treatment with calciumphosphate gel. Following chromatography on hydroxyapatite furtherpurification of anaphylatoxin is carried out in this known process bymolecular sieve chromatography, ion-exchange chromatography onhydroxyapatite, and gel-permeation chromatography. Anaphylatoxin isfinally obtained in crystalline form in a molecularly homogenous,biologically active state.

By the above process, the anaphylatoxin-containing protein preparationobtained by chromatography on hydroxyapatite, which is chemotacticallyactive for neutrophil leucocytes, is separated into two main proteincomponents. The one protein thus obtained has the classical propertiesof anaphylatoxin. For example, in vivo it causes release of histamineand it induces the typical lethal anaphylatoxin shock by contraction ofthe smooth muscles. This substance (classical anaphylatoxin) which isobtained in crystalline form by the known process, however has noappreciable chemotactic activity on neutrophilic leucocytes. Thisactivity is formed by the activation process in crude serum inconcurrence with anaphylatoxin activity, as are other activities.

The second protein, termed cocytotaxin, is likewise obtained by theknown process in a crystalline state; cf. J. H. Wissler, Eur. J.Immunol. 2 (1972), pp. 84-89. The physicochemical properties ofcocytotaxin are similar to anaphylatoxin. But biologically, it isdifferent. Cocytotaxin has no spasmogenic activity of anaphylatoxin.However, as anaphylatoxin, cocytotaxin has no appreciable chemotacticactivity on neutrophilic leucocytes.

Recombination of anaphylatoxin and cocytotaxin in various molar ratiosin vitro leads to a restoration of the chemotactic activity forneutrophil leucocytes intrinsic to contact-activated crude serum. Thus,anaphylatoxin and cocytotaxin together constitute a leucotaxinepreparation as a binary protein system which is biologically active invitro and in vivo. In this system anaphylatoxin is the activityprinciple and cocytotaxin constitutes the activity-inducing cofactor(cochemotaxin). Both separated proteins, anaphylatoxin and cocytotaxin,and the leucotaxine preparation containing the two substances as abiologically active binary protein system are valuable substances with awide array of application possibilities in pathology and immunology. Forexample, they may be used to induce desired focal inflammationprocesses, e.g. in tumours ("biochemical surgery"). Accumulatingleucocytes can be regulated to be composited of selected cell patterns.In addition, by accumulating selective patterns of leucocyte types theycan serve for formation, in statu nascendi, of substances withcell-specific action promoting and inhibiting cell division produced byleucocytes at the reaction site of inflammation. Furthermore, they canbe used to increase, in statu nascendi, the immune status at a reactionsite in tissue, or tumour sites by immunopotentiators secreted byattracted and accumulating leucocyte populations.

In general, purification processes for proteins and other naturalsubstances consist of a sequence of combined separation techniques.Subtle differences in molecular size, charge, form, structure,stability, and nature of the molecule surface between the desirednatural substance and the accompanying foreign substances are used insuch steps for separation. Accordingly, a large number of combinationsof various separation techniques. can be worked out for purification ofa protein. The nature and the conditions of separation steps used, butas well their sequential combination, are of paramount significance foroperational properties, technical practicability, optionalautomatisation possibility and economics of a purification process andfor yield and molecular quality of the natural product investigated.Particular attention has to be focused on optimum form of separationsteps and on their ingenious combination into a purification sequencewithin a frame of structural and functional stability and othermolecular parameters of the substance under investigation. This impliesthat use of identical or similar separation principles (e.g. molecularsieve filtration, dialysis ion-exchange absorption, etc.) in a differentcombination can be of decisive and paramount importance for practice andeconomics of a purification process and yield and quality of the productobtained. In some cases, use or omission of a single technique (e.g.hydroxyapatite chromatography, zone-precipitation chromatography, etc.)at a certain position in the purification sequence, or within a partialsequence, is decisively significant for yield and quality of the desirednatural product as well as for practice and economics of thepurification process. These general relationships and basic principlesinherent to purification processes of natural products are clearlyillustrated e.g. by some well known facts. Thus, within an economicallyand technically operable process for the purification of a naturalproduct, initial dialysis or lyophilization steps are not recommendedprior to reduction of initial volumes of the crude extract by a factorof at least 500-1000 through other techniques.

As above-mentioned, mediators as anaphylatoxin and cocytotaxin are tracesubstances occurring only in very small quantities in the preparedserum. 100 liters of contact-activated serum (corresponding to 250-300liters of blood) contain about 7 to 8 kg of protein in addition to othersubstances. Only about 0.3 to 1.5 g of this protein mass (depending onthe species and on the nature of the contact reaction) representanaphylatoxin, cocytotaxin being present in about twice to three timesthis quantity. Of this, a maximum of 10 to 20% of each protein can beisolated, since any purification is a complex process having onlyrestricted yields. Therefore, a prerequisite for the practical use ofthese proteins is their isolation in appreciable quantities. Hence, verylarge volumes of blood have to be processed.

The above-mentioned process is not suitable for this purpose. Use ofhydroxyapatite for structure-conserving purification of these proteinsis decisively important. For technical and economic reasons, however, itis not possible to chramatograph large volumes of protein solutions onhydroxyapatite columns. Larger amounts of proteins tend to support thestrong tendency of hydroxyapatite to block up. Furthermorehydroxyapatite is very expensive. Its use on large scales is noteconomical. Separation of a part of accompanying foreign proteinsachieved in the known process by absorption on calcium phosphate geladmittedly causes a reduction by about 10% of the amount of foreignsubstance. This reduction is too small to be of practical importance inlarge scale preparation of these mediators. Thus, the residual volume ofprotein solution to be applied on the hydroxyapatite column is still toolarge for economical processing of larger quantities of blood. Accordingto the known process, under optimum conditions, a volume reduction of1000 ml serum to a minimum of some 25 ml of protein solution can beachieved prior to its application onto the hydroxyapatite column.

Another process for preparation of anaphylatoxin has been described byM. Lieflander et al. in Hoppe-Seyler's Z. Physiol. Chem. 353 (1972), pp.385-392. Anaphylatoxin is separated from the foreign proteins byrepeated cation-exchange chromatography and molecular sieve filtrationcombined with lyophilization and dialysis steps. Some of the steps areperformed in organic solvents and in unbuffered acid solution. E. H.Vallota and H. J. Muller-Eberhardt described a modification of thistechniqu-e in J. Exp. Med. 137 (1973), pp. 1109-1123, as did H. N.Fernandez et al. in J. Immunol. 120 (1978), pp. 109-115, in which afurther cation-exchange chromatographic step and an additionalanion-exchange chromatographic step are included. Finally, in J. Biol.Chem. 251 (1979), pp. 6346-6351, C. Gerard and T. E. Hugli suggestedanother modification of this process for isolation of anaphylatoxin, inwhich the activated crude serum was first adjusted to pH 0 (1 mol/lHCl). Without experimental proof, it is implied that theseunphysiological conditions do jeopardize the natural biological qualityof products investigated. They claimed that 65% of the foreign proteinsare precipitated (no experimental data are given). The residual proteinfraction is purified further by a molecular sieve filtration,cation-exchange positive absorption chromatography, and anion-exchangenegative absorption chromatography.

The above-mentioned known processes are also unsuitable for economicalisolation of anaphylatoxin in such quantitiesas are needed for practicalpurposes. The use of large volumes of serum would demand too large andtoo bulky column capacities and almost extremly awkward dialysis andlyophilization steps. In fact, these processes too were conceived andpublished only for use of relatively small initial serum volumes (from0.5 to a maximum of 10 liters of contact-activated serum). Moreover,these processes have the disadvantage that irreversible damage ofprotein structure as a result of the drastic, non-physiologicalconditions (organic, nonaqueous solvents; pH 0) cannot be excluded orare not investigated, also such events are most likely; see for exampleJ. H. Wissler in Proc. Immunosymposium, Vienna 1973, Springer VerlagVienna 1975, pp. 91-105. The isolation of cocytotaxin and the separationof residual trace contaminants of other foreign proteins whichelectrophoretically are hardly detectable, but which arechromatographically obvious, are not envisaged in this method; althoughtheir presence is most likely; cf. H. N. Fernandez at al. (op. cit.) andM. C. Conroy et al. in J. Immunol 116 (1976), pp. 1682-1687.

It is therefore, a primary object of this invention to provide a processfor producing and obtaining anaphylatoxin- and cocytotaxin-containingleucotaxine preparations from large amounts of blood.

It is another object of this invention to provide a process forproducing and obtaining anaphylatoxin and cocytotaxin proteins inmolecularly homegenous form.

It is another object of this invention to provide a process forproducing and obtaining anaphylatoxin- and cocytotaxin-containingleucotaxine preparations and anaphylatoxin and cocytotaxin proteins inbiologically active form.

It is still another object of this invention to provide a process forproducing and obtaining anaphylatoxin-and cocytotaxin-containingleucotaxine preparations and anaphylatoxin and cocytotaxin proteins inappreciable quantities in their native intact structure.

It is still another object of this invention to provide a process forproducing and obtaining anaphylatoxin- and cocytotaxin-containingleucotaxine preparations and anaphylatoxin and cocytotaxin proteins in arelatively simple, automatable and economical manner.

These and other objects and advantages of the present invention will beevident from the following description of the invention.

SUMMARY OF THE INVENTION

The invention is based on a process for producing and obtaininganaphylatoxin- and cocytotaxin-containing leucotaxine preparations andanaphylatoxin and cocytotaxin proteins in molecularly homogeneous,biologically active form from contact-activated mammalian serum by:

separation of the proteins from other serum constituents to obtain aserum protein concentrate fraction,

separation of a part of accompanying foreign blood proteins fromanaphylatoxin and cocytotaxin present in the said protein concentratefraction,

isolation of the leucotaxine preparation by chromatography onhydroxyapatite,

and optionally further purification and/or separation of the .leucotaxine preparation into anaphylatoxin and cocytotaxin proteins bychromatographical methods.

The invention is characterized in that, prior to chromatography onhydroxyapatite, in the above-mentioned process, a major fraction ofaccompanying foreign blood constituents is separated from the said serumprotein concentrate fraction by fractional elution and/or precipitationwith a water-soluble alcohol and/or at least one molecular sievefiltration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 3 show the course of the separation of the crude AT- andCT-containing leukotaxine preparation from the accompanyingcontaminating foreign proteins by a sequence of two preparative and oneanalytical molecular sieve filtration in form of chromatograms.

The isolation of the AT- and CT-containing purified leukotaxinepreparation by chromatography on hydroxyapatite as described in ExampleC1 is shown in the chromatogram of FIG. 4.

The separation of the AT- and CT-containing leukotaxine preparation intothe proteins AT and CT and their further individual purification tomolecular homogeneity as described in Example C5 are shown in thechromatograms presented in FIGS. 5 to 13.

DETAILED DESCRIPTION OF THE INVENTION

The process in accordance with the invention allows processing of largeamounts of serum and thus the fast and economical isolation ofappreciable quantities of anaphylatoxin and cocytotaxin. In accordancewith the invention, the major part of the accompanying foreign proteinsis removed prior to chromatography on hydroxyapatite, which is acritical step for anaphylatoxin and cocytotaxin qualities. Thus, only asmall fraction (in the preferred embodiments less than about 0.09%) ofthe total amount of serum proteins are to be applied to thehydroxyapatite column. A batch can be started for example at least with100 to 200 liters of contact-activated serum (about 300 to 600 liters ofblood). For example, with 100 liters of contact-activated serum, theprotein solution volume still containing all the anaphylatoxin andcocytotaxin can be reduced to less than about 100 to 200 ml, whichrepresents a volume reduction factor of about 500-1000, prior to use ofhydroxyapatite. In addition, the process allows to obtain theinvestigated mediatiors in their native, biologically activeconformation.

The process for production of a leucotaxine preparation containinganaphylatoxin and cocytotaxin and also anaphylatoxin and cocytotaxinproteins will now be specifically explained in detail.

A. PRODUCTION OF ANAPHYLATOXIN AND COCYTOTAXIN IN SERUM. SEPARATION OFAN ANAPHYLYTOXIN AND COCYTOTAXIN-CONTAINING SERUM PROTEIN CONCENTRATEFRACTION FROM THE OTHER SERUM CONSTITUENTS

The production of anaphylatoxin and cocytotaxin in serum, for example bycontact activation, the separation of the serum protein concentratefraction from other serum constituents by aid of a cation-exchanger,elution of the absorbed protein fraction from the ion-exchanger, andconcentration of proteins in the eluate obtained by salting outprecipitation with ammonium sulfate is essentially carried out in theknown manner; cf. J. H. Wissler, Eur. J. Immunol. 2 (1972), pp. 73-83.

Initial, crude material for the isolation of anaphylatoxin- andcocytotaxin-containing leucotaxine preparation and of anaphylatoxin andcocytotaxin (for brevity reasons the substances investigated will bereferred to by the abbreviations AT and CT) in accordance with theprocess of the invention is mammalian serum. Because of its readyavailability, human, bovine, equine, porcine, ovine, canine, feline,rabbit, rat and guinea-pig serum is preferred. The serum is obtained inthe normal way by coagulation of blood and separation of the blood clot,for example by filtration and/or centrifugation, and is incubated forthe formation of AT and CT, for example with dextran, yeast, or with theimmune complex in conventional manner. After a sufficient period ofincubation, for example in case of dextran about 1 h at 37° C., theincubated, contact-activated serum is cooled to a temperature of about0° to 8° C. Then, the insoluble contact substance added for incubationis separated from the contact-activated, AT and CT-containing serum.

The incubated, contact-activated serum containing AT and CT is nowtreated with a cation-exchanger to separate the majority of proteinsfrom other serum constituents. An example of a cation-exchanger suitablefor this purpose are dextrans crosslinked with epichlorohydrin(Sephadex). It is preferable to use a weakly acidic cation-exchangersuch as CM Sephadex C-50 and to perform the treatment at a pH of 4 to 6.To facilitate the charge process, the contact-activated serum can bediluted with a protein-compatible salt solution before treatment withthe cation-exchanger. This salt solution can be used at the same time toadjust the pH. A special example of a salt solution for this purpose isa 0.001 mol/l potassium phosphate-acetate buffer containing 0.2 mol/lNaCl and having a pH of 4.5.

The cation-exchanger is added to the serum in sufficient quantity toabsorb the main protein fraction of serum. As a rule about 1 volume ofswollen ion-exchanger per volume of the serum is sufficient for thispurpose. The supernatent is then separated from the cation-exchangercharged with the proteins, for example by decantation or centrifugation.The charged cation-exchanger is freed from the adhering, negativelyabsorbed serum components by washing with water or a salt solution.Preferably a pH of about 4 to 5 and a maximum temperature of about 15°C. is used. A special example of a salt solution suitable for thewash-out process is the mentioned potassium phosphate-acetate bufferhaving a pH of 4.5.

The protein-charged cation-exchanger is now eluted with aprotein-compatible aqueous salt solution. A salt solution of high ionicstrength with a pH of about 4 to 10 is preferably used for this purpose.Special examples for salt solutions of this kind are aqueous 0.5 mol/lsolution of potassium phosphate of pH 6.5 to 7.5 or a 2 to 5 mol/lsolution of sodium chloride of the same pH.

The eluate obtained containing the bulk of serum proteins and AT and CTis now concentrated prior to subsequent separation of proteins. Thisconcentration process which separates the bulk of aqueous salt solutionfrom the proteins, can be done in various ways. For example, all theproteins can be precipitated by adjusting the eluate to an ammoniumsulfate concentration of about 3.7 mol/l (salting-out precipitation).For this purpose, ammonium sulfate is added in a quantity ofapproximately 630 g/l eluate (saturation about 90%). During thisprocess, the pH is preferably kept at about 4 to 9. The precipitatedproteins are then separated from the almost protein-free supernatent,for example by decantation or centrifugation.

The eluate of the cation-exchange process can also be concentrated byusing other methods, for example ultrafiltration or lyophilization. Butthese processes are time-consuming and relatively expensive. In thiscase for further performance in accordance with the process of theinvention, however, the protein concentrate obtained must likewise beadjusted to an ammonium sulfate concentration of 3.7 mol/l.

In all cases, the AT- and CT-containing protein precipitate formed(serum protein concentrate fraction) is obtained in form of a proteinmud. For purpose of further purification of AT and CT, this mud is usedas such in the manner described below.

B. CRUDE PURIFICATION OF AT AND CT: SEPARATION OF THE BULK OF THEACCOMPANYING FOREIGN PROTEINS OF AT AND CT. PREPARATION OF A AT- ANDCT-CONTAINING "CRUDE LEUCOTAXINE PREPARATION".

In the known process (J. H. Wissler, Eur. J. Immunol 2 (1972), pp.73-83), separation of accompanying foreign proteins and some othersubstances, such as lipids, from AT an CT in the serum proteinconcentrate fraction obtained in step A occurred only to a very smallextent (maximum about 5-10%) by absorption on calcium phosphate gel. Theremaining volume concentrated of protein solution from which the desiredsubstances are to be obtained by chromatography on hydroxyapatite,therefore, is still relatively very large. Thus, only a small quantityof serum can be processed. In addition, the involved dialysis stepnecessary for this purpose is difficult to perform for larger volumes.Additionally some loss of the product occurs.

An outstanding characteristic of the process of the invention is,therefore, the first possibility to separate the major bulk (in thepreferred embodiments more than 99.91%) of the accompanying foreignproteins from AT and CT prior to their chromatography on hydroxyapatite,which is critical for product quality, handling of the purification,process and economics. In this way, a large reduction of the quantity ofprotein to be processed can be achieved prior to this step of theprocess. In accordance with the invention the separation of the foreignproteins from the serum protein concentrate fraction is achieved byfractional elution and/or precipitation with a water-soluble alcoholand/or at least one molecular sieve filtration.

It is possible to remove a considerable amount of accompanying foreignproteins by only one performance of one of the purification methods inaccordance with the invention. Thus, a satisfactory volume reduction ofthe serum protein concentrate fraction is achieved prior to charge ofthe hydroxyapatite column. For example, in the physiological pH rangeabout 30% of the accompanying foreign proteins can be removed byfractional elution, about 60 to 70% by precipitation with awater-soluble alcohol, and up to 90% by molecular sieve filtration.However, as a property of polyelectrolytes, proteins of the serumprotein concentrate fraction tend to adhere very strongly together.Furthermore, no ideal equilibria and distributions are obtained withmacromolecular polyelectrolytes as are proteins. Therefore, for examplein spite of different molecular weights of proteins, by molecular sievefiltration no complete (ideal) separation according to their molecularweight proper is obtained at once. Hence, it is necessary to perform atleast two of the said separation processes in sequence. Thus, preferablythe serum protein concentrate fraction is, for example, firstfractionally eluted and then subjected to a molecular sieve filtration.Or, the foreign proteins are first precipitated with a water-solublealcohol, and, then, a molecular sieve filtration is carried out.Moreover, combinations of fractional elution and/or precipitation with awater-soluble alcohol with at least one preparative and one analyticalmolecular sieve filtration is preferred. All these combinations of thementioned separation steps constitute objects of the invention. It isevident, that certain sequences of separation steps are of lessadvantage than other combinations. Thus, for example, it is imperativeto perform a preparative molecular sieve filtration before an analyticalmolecular sieve filtration: In reverse order of performance difficultiesin handling economics and yield are obvious.

Particularly preferred embodiments of the process in accordance with theinvention consist of the following combinations of separationtechniques:

(a) a fractional elution step followed by two preparative and then oneanalytical molecular sieve filtration;

(b) a fractional elution step followed by one protein precipitation stepwith a water soluble alcohol, then by one preparative, and lastly by oneanalytical molecular sieve filtration;

(c) a protein precipitation step with a water-soluble alcohol followedby one or two preparative and lastly by one analytical molecular sievefiltration.

The performance of the individual separation processes for theseparation of the bulk of the accompanying foreign proteins from the ATand CT contained in the serum protein concentrate fraction in accordancewith the process of the invention will now be described in specificdetail.

Fractional elution of the serum protein concentrate fraction separatesproteins largely independent of their molecular weight. The part of theforeign proteins that is soluble at higher ammonium sulfateconcentrations than AT and CT, is separated from the concentrate. Thisseparation method, therefore, is performed in form of a batch process inconcurrence with the preparation of the serum protein concentratefraction which has the advantage of being rapid and unstricted incapacity. If several separation steps are used in sequence, thefractional elution is, therefore, preferably carried out first.

Before the fractional elution, the protein mixture to be treated must bead]usted to an ammonium sulfate concentration sufficient for theprecipitation of all the proteins. Such a suspension has an ammoniumsulfate content of about 3.7 mol/l (at 0° to 8° C.) i.e. it is saturatedto the extent of 90%. Adjustment to this ammonium sulfate content isdone, for example, at the end of the separation of the entire AT- andCT-containing serum protein eluate from the cation-exchanger.

Concentration of proteins in the cation-exchanger eluate is carried outeither directly by precipitation of proteins with ammonium sulfate. Or,if the eluate had been concentrated by a different process, such asultrafiltration or lyophilization, ammonium sulfate is added to theconcentrate to the given saturation.

For the fractional eluation of proteins, ammonium sulfate concentrationof the serum protein concentrate fraction is adjusted to about 2.6 mol/lby addition of a protein-compatible liquid. Water or, preferably, abuffered salt solution can be used as such a liquid. Preferably, a pHbetween 4 to 8.5 and a temperature of approximately 0° to 8° C. ismaintained. A special example of a suitable salt solution is a 0.001mol/l sodium-potassium phosphate buffer containing 0.1 mol/l NaCl andhaving a pH of 6.3. When such an (ammonium sulfate-free) salt solutionor water is used, about 0.4 volume parts per volume part of the serumprotein concentrate fraction (as protein mud) is necessary to achievethe desired reduction in the ammonium sulfate concentration.

The ammonium sulfate concentration of the obtained protein suspensionshall not be much lower than about 2.6 mol/l. Otherwise the AT and CTare dissolved as well. On the other hand, adjustment to an ammoniumsulfate concentration much higher than about 2.6 mol/l is notadvanteous, because then less foreign proteins are dissolved and thus,efficiency of the fractional elution decreases. To obtain a favourablesolution equilibrium at the given ammonium sulfate concentration, theprotein suspension is maintained at the given temperature and pHconditions for some time, preferably about 10 to 24 h with stirring. Theremaining protein precipitate is then separated from the supernatant,for example by decantation or centrifugation.

Fractional elution in principle is the reverse process of fractionalprecipitation. For this reason this purification step, in principle, maybe carried out as fractional precipitation. In such a case, the eluateof cation-exchange process is adjusted to an ammonium sulfateconcentration of only about 2.6 mol/l instead of about 3.7 mol/l. Theprecipitating AT- and CT-containing protein fraction is separated fromthe soluble supernatant. However, as is known from principles in proteinfractionations, due to kinetics of precipitation equilibria, fractionalelution has selective advantage over fractional precipitation asseparation process. It may give higher yields than fractionalprecipitation. This also applies to this AT and CT fractionationprocess.

Separation of a part of accompanying foreign proteins from AT and CT byprotein precipitation with a water-soluble alcohol makes use of the factthat under certain conditions a fraction of foreign proteins, but not ATand CT, is precipitated upon addition of a water-soluble alcohol to theprotein solution. As for a fractional elution, this proteinprecipitation with alcohol is largely independent of the molecularweight of proteins. A large part of the accompanying proteins isseparated from AT and CT. This separation step is preferably performedafter the fractional elution process or instead of the latterpurification step. In this way, for the following molecular sievefiltration the charge of the column used is considerably reduced.

Treatment with alcohol is carried out at a pH of about 3.5 to 5.5 andpreferably at about 4.0, and at a maximum temperature of 8° C. andpreferably about 5° C. Maintenance of given pH range is crucial. Atlower pH, isolated proteins may be damaged. Higher pH may lowerefficiency of the precipitation step. However, adjusting the pH only tothe given range does not bring about precipitation of the foreignproteins.

The water-soluble alcohol is added in amounts of about 180 to 250, andpreferably about 200 volume parts per 1000 parts of the proteinsolution. Smaller quantities of alcohol do not achieve completeprecipitation. Higher alcohol concentrations not induce betterprecipitating effects. They may, however, increase the risk for damageof mediators investigated by altering their confirmation. Specialexamples of suitable water-soluble alcohols are methanol, e thanol,propanol, and ethylene glycol. Ethanol is preferred as the water-solublealcohol.

Protein precipitation with alcohol is performed with the(ammonium-sulfate-containing) muddy serum protein concentrate fractionobtained from the eluate of cation-exchange process or with the muddyprotein residue obtained by the fractional elution step after theirsolution in a minimum volume of a protein-compatible liquid. A specialexample of a suitable solution is 0.01 mol/l ammonium acetate solutioncontaining 0.2 mol/l NaCl and having a pH of 5.0. The pH of the solutionis then adjusted to the given range for example with glacial acetic acidafter which the alcohol is added. For precipitation of foreign proteins,the solution is then maintained under the given conditions for sometime, preferably with stirring. The precipitated foreign proteins arethen separated from the supernatant which contain AT and CT dissolved,for example by decantation or centrifugation. The alcohol can then beremoved, for example by dialysis, ultrafiltration, or lyophilization,from the supernatant protein solution. If a molecular sieve filtrationstep follows for further purification of AT and CT, the alcohol (46daltons for C₂ H₅ OH) may be directly separated from the AT and CT (ca.10,000 daltons) without the above-mentioned auxiliary methods.

Molecular sieve filtration achieves separation of proteins according totheir molecular weights. Since the bulk of the foreign proteins havemolecular weights greater than AT and CT, they can be separated off inthis manner. A hydrophilic water-swelling molecular sieve is used forseparation of the proteins by molecular weight. Examples of suitablemolecular sieves are dextrans cross-linked with epichlorohydrin(Sephadex), agaroses cross-linked with acrylamide (Ultrogels), andthree-dimensionally cross-linked acrylamides (Biogels), whose exclusionlimits are higher than the separation limits used.

If several separation steps are used, the molecular sieve filtration ispreferably carried out after a fractional elution and/or proteinprecipitation with a water-soluble alcohol. In this way a major fractionof foreign proteins from all molecular weight ranges is already removedby the preceding separation steps. The proteins mass to be removed bythe molecular sieve step is thus already considerably smaller. Dependingon length-to-diameter ratio of the column used and particle diameter ofthe gel matrix, molecular sieve filtration is termed "preparative" or"analytical". A molecular sieve filtration is preparative when thechromatography is performed on columns with a length-to-diameter ratioof up to 10:1 and a charge of column of up to 1/3 of its capacity or useof total separation volume of the matrix. An analytical molecular sievefiltration means a length-to-diameter ratio greater than 10:1, andpreferably about 50:1, and a maximum charge of column of up to 3% of itscapacity.

In preparative molecular sieve chromatography, gel matrices with thelargest possible particle size are used for maximum flow-through ratesof mostly viscous protein solutions at reasonably low pressures. Inanalytical molecular sieve filtration the particle size ranges of thegel matrix selected are as small as possible, to obtain a maximum numberof theoretical plates, a flow rate of the mobile phase equal to 2-4 cm/hcombined with a pressure which is limited to technical and safetyaspects. These parameters are dependent on the structure of the gelmatrix and may vary from gel to gel.

If several preparative molecular sieve filtrations are performed insequence, graduated separation limits can be selected. For example, aseparation limit of 20,000 daltons can be used in a first filtrationstep and of 13,000 daltons in a second. This can be followed by ananalytical molecular sieve filtration with upper and lower separationlimits of 11,000 and 6000 daltons. The exclusion limit of the gel usedin all cases must be higher than about 10,000 daltons to allow a volumedistribution of AT (molecular weight about 9500) and CT (molecularweight about 8500) between the stationary gel matrix phase and themobile aqueous buffer phase. Guinea-pig AT has a somewhat highermolecular weight of about 14,000 daltons. Therefore, in processingguinea-pig serum correspondent higher separation and exclusion limitsmust be used.

The "exclusion limit" is a hydrodynamic parameter of a dissolvedparticle corresponding to the pore size of the gel matrix. Particleswith a greater hydrodynamic parameter cannot penetrate the gel matrix(volume distribution coefficient K_(D) =0). The "separation limit"refers to a hydrodynamic parameter which has been chosen for theseparation of dissolved particles from others, with values between thevolume distribution coefficients K_(D) =0 and K_(D) =1.

For molecular sieve filtration the proteins are applied to the molecularsieve after solution in a protein-compatible liquid. A special exampleof a suitable solvent is 0.01 mol/l sodium-potassium phosphate solutioncontaining 0.3 mol/l NaCl and having a pH of 6.5. After the filtrationAT- and CT-containing fractions are collected. The dissolved proteins ofthe fractions are preferably concentrated by addition of ammoniumsulfate to a concentration of 3.7 mol/l (90% saturation).

After separation of the precipitated proteins form the supernatant theyare again dissolved in a protein-compatible liquid and, if necessary,subjected to a further purification step.

Between the above-described purification steps, if necessary, proteinsolutions can be separated from salts by dialysis or ultrafiltration,e.g. against sodium-potassium phosphate buffer. By selecting anappropriate mobile phase in the usual way, a modified molecular sievefiltration can also be used for this purpose. In the molecular sievefiltrations about 0.4 mol/l ammonium sulfate is preferably added to theprotein solution. In contrast to higher concentrations of this salt, atthis concentration ammonium sulfate exerts strong salting-in effect onproteins. Thus, proteins are better kept in solution during themolecular sieve filtration. Moreover, ammonium sulfate prevents growthof microorganisms and inhibits certain enzymes. Hence, it contributes tostabilization of the AT and CT, which is important when chromatographyis performed at higher temperature (above about 20° C.) and undernon-sterile conditions. To prevent oxidation, about 0.001 mol/l ofcysteine is preferably added to protein solutions throughout.

The temperature and pH conditions during molecular sieve filtration arenot particularly critical. If the native conformation of the proteinsshall be preserved, a temperature range of about 0° to 8° C. andpreferably about 0° to 4° C. is optional. The preferred pH range isbetween 6 and 9.

Use of above-mentioned separation processes according to the inventions,achieves separation of a major part of the accompanying foreign proteinsfrom the mediators to be isolated, AT and CT, already prior tochromatography on hydroxyapatite. For example, according to one of thepreferred embodiments, using fractional elution, two preparative and oneanalytical molecular sieve filtrations in sequence, a volume of proteinsolution of 100 liters of initial crude contact-activated serum(corresponding to about 35 liters of wet protein mass, or 7-8 kg of dryprotein mass) can be reduced to less than about 100 to 200 ml before thefirst purification step (chromatography on hydroxyapatite). It isobvious that this makes possible for the first time preparing largeramounts of AT and CT. The quantity of protein to be chromatographed onhydroxyapatite is relatively small and already enriched in AT and CT.Hydroxyapatite columns with total absorption capacity for theintermediate product thus obtained ("crude leucotaxine preparation")from up to 200 liters of contact-activated crude serum can still beprocessed within the laboratory.

(C) ISOLATION OF THE AT- AND CT-CONTAINING HIGHLY PURIFIED LEUCOTAXINEPREPARATION BY CHROMATOGRAPHY ON HYDROXYAPATITE. FURTHER PURIFICATION OFTHE PREPARATION AND ITS SEPARATION INTO ANAPHYLATOXIN AND COCYTOTAXINPROTEINS

After performance of one or of a combination of the separation processesdescribed in section B) the resulting protein solution, in which AT andCT are already enriched, is chromatographed on hydroxyapatite. Thisseparates other foreign proteins and leads to an AT- and CT-containinghighly purified leucotaxine preparation. The process is preferablycarried out in the manner described by J. H. Wissler in Eur. J. Immunol.2 (1972) p. 76, right-hand column.

Ammonium sulfate and other salts, especially phosphate in theconcentrated solution from the last preceding separation step (crudeleukotaxine preparation) must be removed, preferably by dialysis, priorto application of protein solution to the hydroxyapatite. Apart fromviscosity increase, however, only the phosphate concentration of theprotein solution is critical for the chromatography on hydroxyapatite.The AT and CT are eluted by a sodium-potassium phosphate concentrationgradient, which preferably is linear. The AT- and CT-containingfractions are collected and then concentrated, e.g. by addition ofammonium sulfate to a concentration of 3.7 mol/l and separation of theprecipitated proteins by usual methods.

The leukotaxine preparation obtained by chromatography on hydroxyapatiteconsists of about 600 mg protein from 100 liters of serum. In general itcontains about 60 to 70% of AT and CT. Thus, AT and CT constitutealready a major portion in this purified product. However, if necessary,it may be further purified from residual contaminations by applicationof additional purification steps. Suitable further separation steps arerechromatography on hydroxyapatite, zone-precipitation chromatography,analytical molecular sieve filtration, or combinations of these steps.

The rechromatography on hydroxyapatite can be performed in the mannerdescribed by J. H. Wissler in Eur. J. Immunol. 1 (1972), p. 77, (rightpanel). Any foreign salts and ammonium sulphate possibly present areremoved first from the leukotaxine preparation, preferably by dialysis.The resulting solution of the leukotaxine preparation then is applied tothe hydroxyapatite column and eluted by a sodium potassium phosphateconcentration gradient. The fractions containing the leukotaxinepreparation are collected and concentrated in the usual manner.

In the zone-precipitation chromatography (cf. J. Porath, Nature, 196(1962), p. 47-48), residual protein contaminations in the leukotaxinepreparation are separated off by salting-out fractionation of theproteins with a salt concentration gradient. Temperature and pH, columndimensions, type of the salt, shape of the gradient, and column chargecan be varied within relatively wide limits.

The temperature for zone-precipitation chromatography can be between 0°and 40° C. Preferably, a temperature range from about 0° to 10° C. isused, especially from about 4° to 6° C. The pH can be between 4 and 10;preferably, a pH range of 6 to 8 is used, especially a pH of about 7.The length-to-diameter ratio of the column used should be greater thanabout 10:1. A ratio of 30 to 100:1 and especially of about 50:1 ispreferred. All salts having salting-out properties for proteins andbeing protein-compatible are suitable. Examples of such salts are sodiumpotassium phosphate, ammonium sulfate, and sodium sulfate. Ammoniumsulfate is preferred.

The salt concentration gradient can have any desired shape within aslong as the salting-out criteria of proteins provide protein separation.Linear concentration gradients are preferred, especially an ascendinglinear concentration gradient from 25 to 80% ammonium sulfatesaturation. The maximum column charge is about 5% and preferably about1% of total column volume.

Analytical molecular sieve filtration for further purification of theleukotaxine preparation can be performed in the same manner as describedfor separation of foreign proteins prior to chromatography onhydroxyapatite. The same molecular sieves, columns and performanceconditions can be used.

A considerable separation of contaminating foreign proteins stillpresent in the leukotaxine preparation (in addition to AT and CT) canalready be achieved by one of the above-mentioned, additionalpurification steps. For example, by rechromatography on hydroxyapatite,a leukotaxine preparation is obtained in which AT and CT make up toabout 91% of total protein present. Zone-precipitation chromatographyincreases the AT and CT fraction of the leucotaxine preparation to about94%, and a single analytical molecular sieve filtration increases it toabout 95%. If only one additional purification step is performed,zone-precipitation chromatography is preferred.

The basic principle of separation of proteins by zone-precipitationchromatography are different, structure-related reversible solubilitycharacteristics of proteins. They belong to the most sensitive molecularseparation criteria and are often used for demonstration of molecularhomogeneity of a protein. This explains preference of zone-precipitationchromatography over the various types of rechromatography methods, inwhich purification effects are based on optimum approximation to idealequilibria in non-ideal polyelectrolyte systems.

For therapeutic use, the leukotaxine preparation is preferably almostcompletely freed from contaminating foreign proteins by combination ofat least two of the mentioned purification steps. Especially, a sequenceof steps is preferred, in which the leukotaxine preparation obtained bychromatography on hydroxyapatite is first rechromatographed onhydroxyapatite, then subjected to a zone-precipitation chromatography,and finally to an analytical molecular sieve filtration. In anotherpreferred embodiment rechromatography on hydroxyapatite can also beperformed after the zone-precipitation chromatography. In thesepreferred embodiments a leukotaxine preparation is obtained in whichtotal protein content consists of up to more than 99% of AT and CT.

The AT and CT-containing leukotaxine preparation obtained bychromatography on hydroxyapatite can be separated by chromatographicmethods into the individual components investigated, AT and CT, eitherdirectly after the first chromatography on hydroxyapatite, or after oneof the above-mentioned additional purification steps. If necessary, ATand CT can then be purified individually from small amounts ofaccompanying foreign proteins. This is achived with the purificationtechniques above-mentioned for purification of the leukotaxinepreparation. Thus, AT and CT can be obtained in crystalline form.

The resolution of the leukotaxine preparation into AT and CT ispreferably carried out by modification of the process described by J. H.Wissler in Eur. J. Immunol. 2 (1972), p. 78, using cascade and recyclingmolecular sieve filtration. Separation into the two proteins occurs inthe first of the two separation steps. The second step serves forremoval of small amounts of the respective other protein from the AT andCT respectively.

The cascade and recycling molecular sieve filtration can be performedunder the conditions described above for the analytical molecular sievefiltration. The same molecular sieves and the same chromatographyconditions can be used. Sephadex G50 is preferred, with a column of aminimum length-to-diameter ratio of about 50:1 and a maximum charge upto about 3% of the column capacity. The solvents necessary for elutionpreferably are the same as used for analytical molecular sievefiltration.

In cascade molecular sieve filtration, at a certain separation limit,the eluate is applied to a column cascade composed, for example, of twocolumns of the same specifications. Through increased column length, theleukotaxine preparation is separated into two main fractions of amolecular weight of 9500 daltons (AT) and 8500 daltons (CT).

After separation, both fractions (AT and CT) are individuallyconcentrated e.g. by salting-out precipitation with ammonium sulfate.For separation of the residual component of each of the other protein,AT and CT can be subjected separately to a further cascade or recyclingmolecular sieve filtration. The latter differs from the former in thatthe eluate is not passed, at the given separation limit, through acascade of further columns but is reapplied to the same column. Thecolumn length for the proteins is thus increased. The small amounts ofeach of the protein isolated from the bulk of the other protein then canbe added to the main fraction obtained in the first cascade molecularsieve filtration.

As already mentioned, the separation of the leukotaxine preparation intoits protein components can be carried out directly after having beenyielded in the first chromatography on hydroxyapatite or following oneof the described additional purification steps. If separation into ATand CT is intended, it is preferably performed following the firstchromatography on hydroxyapatite. Effective purification of the AT andCT proteins obtained is achieved by individual sequences consisting ofrechromatography on hydroxyapatite, zone-precipitation chromatography,and/or analytical molecular sieve filtration.

As already stated, temperature and pH conditions are not particularlycritical in most of the steps of the process in accordance with theinvention. However, if the leukotaxine protein preparation or the AT andCT proteins have to be obtained in their native conformation, theseparation and purification steps are carried out under almostphysiological pH and salt conditions, and preferably at low temperatureswith a maximum of about 10° C. and preferably 6° C. An essentialadvantage of the process in accordance with the inventions is that theseconditions can be easily verified.

The resulting leukotaxine protein reparation or the separated AT and CTproteins can be stored in a physiological, buffered salt solution, e.g.in 0.0015 mol/l sodium-potassium phosphate solution containing 0.15mol/l (0.9 w/v %) NaCl, 0.001 mol/l cysteine and having a pH of 7.4.After usual sterilization by filtration (pore size 0.2 μm), the proteinpreparation remains native and biologically active at room temperature(for at least 200 h) or frozen at -25° C. (for at least 6 years). Thisstability of the protein preparations can be considered as one of thecriteria of molecular homogeneity. A safe storage form at temperaturesbetween -20° and +50° C. is the crystalline state of the proteins in theform of a crystal suspension, prepared according to the method describedby J. H. Wissler, op. cit. In this form it is also protected againstinfection and degradation by microorganisms.

The invention will now be given in detail by examples describing theisolation of the leucotaxine preparation and of the individual AT and CTproteins starting from porcine blood. However, in terms of the speciesused the invention is not restricted to this form of embodiments.

EXAMPLE A Production of Anaphylatoxin and Cocytotaxin in serumSeparation of an AT- and CT-containing serum protein concentratefraction from other serum constituents

The production of AT and CT in the serum and the separation of a majorserum protein concentrate fraction from the other serum constituents areexplained.

300 liters of porcine blood are coagulated without additives at roomtemperature. The supernatant serum is separated from the clot bycentrifugation. The clear serum is then mixed and contact-activatedimmediately with 5 mg dextran per ml serum or with 100 mg bakers' yeastper ml serum and incubated by stirring in a water bath for 1 h at 37° C.Then, the mixture is cooled to 4° C. and dextran or yeast is removed bycentrifugation. The presence of anaphylatoxin and of cocytotaxine in the120 liters of contact-activated serum obtained, which contains about 9.2kg of protein, is demonstrated. by activity assays (chemotaxis ofneutrophil leukocytes and anaphylatoxin activity).

Unless otherwise specified, the following purification techniques arethen performed in the presence of 0.001 mol/l cysteine at a temperatureof 0° to 8° C.:

120 liters of contact-activated serum are adjusted to pH 4.5 with 5mol/l acetic acid and mixed with 240 liters of 0.001 mol/ltivated serumare adjusted to pH 4.5 with 5 mol/l acetic acid and mixed with 240liters of 0.001 mol/l potassium phosphate-acetate buffer containing 0.2mol/l NaCl and having a pH of 4.5. Then, 3 volume parts of the proteinsolution obtained are mixed by stirring in 1 volume part of a swollen,regenerated cation-exchanger (Na⁺ as mobile exchangeable counter-ion)coupled to a dextran matrix cross-linked by epichlorohydrin (CM-SephadexC-50). The ion-exchanger used has been pretreated for at least 1 daywith porcine serum for surface inactivation, regenerated prior to use,and equilibrated with the phosphate-acetate buffer of pH 4.5.

The resulting mixture is stirred for about 24 h. The ion-exchange gel isthen settled and separated from the supernatant by filtration on aBuchner funnel. The gel is washed with three 120-liter portions of 0.001mol/l potassium hydrogen phosphate-acetate buffer containing 0.2 mol/lNaCl and having a pH of 4.5, until the extinction of the filtrate at 280nm is E=1.0.

For elution of the absorbed proteins, the charged ion-exchange gel issuspended three times in the same volume part of 0.5 mol/l potassiumphosphate buffer at pH 6.5. The ion-exchange gel is separated from thesolution each time by centrifugation or filtration. The proteinsolutions obtained are combined (360 liters).

The combined eluates of the cation-exchange gel are adjusted to aconcentration of ammonium sulfate of 90% saturation by addition of 630 gof ammonium sulfate per liter eluate protein solution. During addition,the pH of the protein solution is checked continuously and maintainedbetween 6.5 and 7.0 by addition of 2 mol/l ammonia. AT and CTprecipitate of the solution together with the majority of serumproteins. The protein precipitate is separated from the almostprotein-free supernatant salt solution by centrifugation for 1 h at10,000×g. 49 liters of ammonium sulfate-containing protein mud areobtained, containing about 3.5 kg of protein (serum protein concentratefraction).

EXAMPLE B Crude purification of AT and CT: Separation of the bulk of theaccompanying foreign proteins from the AT and CT. Preparation of a crudeAT- and CT-containing leukotaxine preparation EXAMPLE B1 Crudeseparation of AT and CT by fractional elution of contaminating foreignproteins

For fractional elution, 49 liters of the serum protein concentratefraction obtained as a u:d as described above under example A, having anammonium sulfate concentration of about 3.7 mol/l (90% saturation) aretreated with 0.00 mol/l sodium-potassium phosphate buffer containing 0.1mol/l NaCl, 0.001 mol/l cysteine and having a pH of 6.3 in quantities of0.4 volume parts per volume part of serum protein concentrate fractionmud under stirring for about 24 h. Then, the residual insoluble proteinprecipitate which contains almost all AT and CT, is separated from thesupernatant eluate by centrifugation at 10,000×g. About 70% (2.7 kgprotein) of the starting serum protein concentrate fraction remain inthe form of a mud (volume: about 14 liters). For removal of ammoniumsulfate, this protein mud containing AT and CT and representing a crudeleukotaxine preparation is dialysed at a membrane with an exclusionlimit of 1000 daltons against 0.001 mol/l sodiumpotassium phosphatesolution containing 0.1 mol/l NaCl, 0.001 mol/l cysteine and having a pHof 7.4. 40 liters of ammonium sulfate-free protein solution are obtainedand applied to a hydroxyapatite column according to Example C.

EXAMPLE B2 Crude Separation of AT and CT By Fractional Precipitation ofContaminating Foreign Proteins

For fractional precipitation, 49 liters of the serum protein concentratefraction obtained as a mud as described above under Example A, aredissolved in a minimum volume of 0.01 mol/l ammonium acetate buffercontaining 0.2 mol/l NaCl and having a pH of 5.0. One volume part of theresulting protein solution is adjusted to pH 4.0 with glacial aceticacid under stirring and mixed with 0.2 volume parts of 96% ethanol at atemperature of 0° C. During addition of ethanol, the temperature and thepH are maintained constant. The mixture is stirred for 1 h at 0° C. 70%(2.45 kg) of foreign proteins present in solution are precipitated andseparated from AT- and CT-containing supernatant solution bycentrifugation for 1 h at at least 16,000× g. The separated precipitateof foreign proteins (10 liters) is washed four times with an equalvolume of 0.01 mol/l ammonium acetate buffer containing 0.2 mol/l NaCl,200 ml ethanol per liter buffer and having a pH of 4.0, at a temperatureof 0° C. The washings are combined with the AT- and CT-containingsupernatant solution from the centrifugation and the mixture is adjustedto pH 5.0 using 2 mol/l ammonia. Then, the ethanol is separated off bylyophilization, molecular sieve filtration, or dialysis (exclusion limit1000 daltons) against the ammonium acetate buffer. If dialysis ormolecular sieve filtration have been used, the proteins are precipitatedin the alcohol-free solution obtained by the addition of ammoniumsulfate (90% saturation) and separated from the almost protein-freesupernatant salt solution by centrifugation at 10,000×g. The proteinresidue obtained by lyophilization or precipitation (1.05 kg protein)which contains AT and CT and represents a crude leukotaxine preparation,is freed from the ammonium sulfate and other salts by dialysis as givenin Example B1. The protein solution obtained can be applied onto thehydroxyapatite column as an ammonium sulfate-free solution (volume 16liter) as described in Example C2.

EXAMPLE B3 Crude Separation of AT and CT From Contaminating ForeignProteins by Preparative Molecular Sieve Filtration

For preparative molecular sieve filtration 1 volume part (49 liters) ofserum protein precipitate fraction obtained as a mud as described aboveunder Example A, are dissolved in 2 volume parts of 0.03 mol/lsodium-potassium phosphate solution containing 0.3 mol/l NaCl, 0.001mol/l cysteine and having a pH of 6.7. The solution is centrifuged for30 min at 4° C. and 10,000×g. for removal of small amounts of insolubleparticles. Then, the clear solution (147 liters) is subjected to apreparative molecular sieve filtration. Therefore, it is applied to acolumn packed with a molecular sieve matrix of dextran cross-linked withepichlorohydrin (Sephadex G-50; particle size 50 to 150 μm). The columnhas a 10-fold volume of the protein solution volume and alength-to-diameter ratio of 10:1. The column is eluted with ascendingflow (rate 3 cm/h) with 0.03 mol/l sodium-potassium phosphate buffercontaining 0.3 mol/l NaCl, 0.001 mol/l cysteine, 0.4 mol/l ammoniumsulfate and having a pH of 6.7. The amount of buffer used for elutioncorresponds to the total column volume. The eluate is divided into twofractions with a separation limit of 20,000. The fraction with molecularweight larger than 20,000 daltons contains 85% of the initial foreignprotein mass. The AT- and CT-containing fraction (0.5 kg of protein)with molecular weights smaller than 20,000 daltons is collected andconstitutes the crude leukotaxine preparation. The proteins of thisfraction are concentrated as given in Example B2 by precipitation withammonium sulfate and, then, separation of ammonium sulfate by dialysis.The resulting solution which still contains 15% of the original proteinmass (7.5 liters), has a volume of 22.5 liters and can be applied to thehydroxyapatite column as will be described in Example C.

EXAMPLE B4 Crude Separation of AT and CT From Contaminating ForeignProteins by a Sequence of Fractional Elution and Preparation MolecularSieve Chromatography

49 liters of the serum protein concentrate fraction obtained as a mud asdescribed above in Example A, are fractionally eluted as given inExample B1. The resulting 14 liters of protein mud containing AT and CT,are dissolved in sodiumpotassium phosphate buffer as described inExample B3. The resulting solution is processed for chromatography onSephadex G-50 as described in Example B3. The fraction with molecularweights smaller than 20,000 daltons is collected, concentrated withammonium sulfate, and finally dialysed. 6.0 liters ofammonium-sulfate-free protein solution is obtained, with a proteincontent of 0.4 kg, containing AT and CT and constituting a crudeleukotaxine preparation. This solution can be further processed as givenin Example C.

EXAMPLE B5 Crude Separation of AT and CT From Contaminating ForeignProteins by a Sequence of Fractional Precipitation and PreperativeMolecular Sieve Chromatography

49 liters of the serum protein concentrate fraction obtained as a mud asgiven in Example A are treated with ethanol for fractional precipitationof foreign proteins as given in Example B2. The protein residue obtainedafter removal of ethanol and concentration of proteins from thesupernatant solution (1.05 kg of protein) then is dissolved andchromatographed on a molecular sieve as given in Example B3. 2.3 litersof ammonium-sulfate-free protein solution are obtained, with a proteincontent of 160 g, containing AT and CT and representing a crudeleukotaxine preparation.

EXAMPLE B6 Crude Separation of AT and UT From Contaminating ForeingProteins by a Sequence of Fractional Elution, Two Preparative and OneAnalytical Molecular Sieve Filtrations

49 liters of the serum protein concentrate fraction obtained as mud asdescribed above in Example A are fractionally eluted and then subjectedto a preparative molecular sieve filtration as described in Example B4.

After concentration of proteins in the AT- and CT-containing fractionwith a molecular weight smaller than 20,000 daltons by salting-outprecipitation with ammonium sulfate, the obtained AT- and CT-containingprotein precipitate is dissolved in 0.03 mol/l sodium-potassiumphosphate buffer containing 0.3 mol/l NaCl, 0.001 mol/l cysteine andhaving a pH of 6.7. 1.5 volume parts of buffer per volume part ofprotein precipitate mud is used. A small amount of residual insolublematerial is centrifuged off. For preparative molecular sieverechromatography the solution is applied to a column packed with amolecular-sieve matrix of dextran cross-linked with epichlorohydrin(Sephadex G-50; particle size 50 to 150 μm). The column has a 10 foldvolume of the protein solution and a length-to-diameter ratio of 10:1.The column is eluted in ascending mode at 3 cm/h with 0.03 mol/lsodium-potassium phosphate buffer containing 0.3 mol/l NaCl, 0.001 mol/lcysteine, 0.4 mol/l ammonium sulfate and having a pH of 6.7. Theresulting eluate is divided into three fractions with separation limitslarger than 20,000, smaller than 13,000 daltons and an intermediatefraction. The AT- and CT-containing protein fraction with a molecularweight range smaller than 13,000 daltons contains 130 g of protein andrepresents a crude leukotaxine preparation. It is adjusted to anammonium sulfate concentration of 3.7 mol/l for salting-outprecipitation of proteins. The protein-precipitate is separated from thesupernatant by centrifugation.

The AT- and CT-containing protein precipitate obtained is dissolved in1,5 volume parts of the buffer solution as described above in thepreceding molecular sieve filtration with ammonium sulfate omitted. Thesolution is centrifuged for 1 h at 10,000×g to remove a small quantityof insoluble residue.

For analytical molecular sieve filtration, the obtained clear solutionis applied to a column packed with a molecular-sieve matrix of dextrancross-linked with epichlorohydrin (Sephadex G-50) with a particle sizeof 20 to 80 μm. The column used has a 50-fold volume of the proteinsolution and a length-to-diameter ratio of 50:1. For elution inascending mode (3 cm/h) a 0,03 mol/l sodium-potassium phosphate buffercontaining 0.3 mol/l NaCl, 0.001 mol/l cysteine, and 0.4 mol/l ofammonium sulfate with a pH of 6.7 is used. The resulting eluate isdivided into several fractions, which chemotactic properties forneutrophil leukocytes and anaphylatoxin activity are tested. Anintermediate fraction with a molecular weight range of 11,000 to 6000daltons, containing essentially all AT and CT and representing a crudeleukotaxine preparation is separated off. This fraction is adjusted toan ammonium sulfate concentration of 3.7 mol/l for salting-outprecipitation of proteins. The precipitated proteins are removed fromthe supernatant by centrifugation. They can be dissolved as usuallydescribed in the preceding steps or, preferentially, as given in ExampleC1, and a small amount of insoluble material is removed bycentrifugation. Yield: 200 ml of clear concentrated protein solutionwith a protein content of 0.9 g.

FIGS. 1 to 3 show the course of the separation of the crude AT- andCT-containing leukotaxine preparation from the accompanyingcontaminating foreign proteins by a sequence of two preparative and oneanalytical molecular sieve filtration in form of chromatograms.

EXAMPLE B7 Crude Separation of AT and CT From Contaminating ForeignProteins by a Sequence of Fractional Elution, Fractional Precipitation,One Preparative and One Analytical Molecular Sieve Filtration Processes

49 liters of the serum protein concentrate fraction obtained as a mud asdescribed in Example A, are treated sequentially for fractional elution(Example B1; result: 14 liter of protein mud) and for fractionalprecipitation of foreign proteins (Example B2). The protein residueobtained (1.05 kg of protein) after removal of ethanol and concentrationof soluble proteins of the supernatant solution is subjected to thesecond preparative molecular sieve filtration and, then, sequentially toan analytical molecular sieve filtration, both described in Example B6.The same result for the crude, AT- and CT-containing leukotaxinepreparation as in Example B6 is obtained.

EXAMPLE B8 Crude Separation of AT and CT From Contaminating ForeignProteins by a Sequence of Fractional Precipitation, Two Preparative andOne Analytical Molecular Sieve Filtration Processes

49 liters of the serum protein concentrate fraction obtained as a mud asdescribed in Example A, are first treated sequentially with ethanol forfractional precipitation of foreign proteins and subjected to apreparative molecular sieve filtration as described in Example B5. Theprotein residue obtained after concentration (160 g of protein) isdissolved as described and then subjected as given in Example B6 to asequence of the second preparative and to the analytical molecular sievefiltration. The same result for the crude, AT- and CT-containingleukotaxine preparation as in Example B6 is obtained.

EXAMPLE C Isolation of the AT- and CT-Containing Purified LeukotaxinePreparation by Chromatography on Hydroxyapatite. Further Processing to aHighly Purified Leukotaxine Preparation into Separated, Individual ATand CT Proteins and to Their Molecular Homogeneity EXAMPLE C1Chromatography of the AT- and CT-Containing Crude LeukotaxinePreparation B6 on Hydroxyapatite

The AT- and CT-containing crude leukotaxine protein precipitate obtainedin Example B6 is dissolved in a minimum volume of 0.0015 mol/lsodium-potassium phosphate buffer containing 0.15 mol/l NaCl and 0.001mol/l cysteine. A small amount of residual insoluble material isdiscarded after separation by centrifugation (10,000×g,1 h, 4° C.). Theclear solution is dialysed against 0.001 mol/l sodium-potassiumphosphate buffer containing 0.1 mol/l NaCl, 0.001 mol/l cysteine andhaving a pH of 7.4, ultrafiltered, or desalted by molecular sievefiltration (exclusion limit: 1000 daltons) until no sulfate is any moredetectable in the solution. A small fraction of insoluble proteins isthen removed by centrifugation for 1 h at 10,000 g and 4° C.

The clear AT- and CT-containing leukotaxine protein solution obtained(200 ml; protein content: 9.0 g) is applied to a column packed withhydroxyapatite. The column has a length-to-diameter ratio of 10:1 and3-fold volume of the protein solution volume to be applied (45 mgprotein/ml). Prior to the application of the leukotaxine preparation,the column is first equilibrated with a 5-fold volume of 0.001 mol/lsodium-potassium phosphate buffer containing 0.1 mol/l NaCl, 0.001 mol/lcysteine and having a pH of 7.4 (flow rate 3 cm/h).

The negatively adsorbing proteins are washed out by elution with thebuffer solution used to equilibrate the column. Then, elution of the AT-and CT-containing leukotaxine fraction is performed within 4 days with alinear phosphate concentration gradient of 0.001 mol/l to 0.5 mol/l ofsodium-potassium phosphate buffer of pH 7.4, having the given constantNaCl and cysteine concentrations. The elution gradient is measured byconductivity. A usual step (salting-out precipitation orultrafiltration) for concentration of active fractions is applied. Asuitable buffer for dissolving the protein concentrate is 0.001 mol/lsodium-potassium phosphate buffer containing 0.001 mol/l cysteine andhaving a pH of 7.2. Thus, it is possible to adjust to any other saltconcentration by addition of a small volume of a concentrated saltsolution. For example, if required, addition of 0.05 volume parts of a 2mol/l NaCl solution of pH 7.2 results a phosphate buffered proteinsolution with 0.1 mol/l NaCl. A protein solution volume of 14 ml isobtained, containing 650 mg of the product, which represents aleukotaxine preparation with an AT and CT content of about 70% of totalprotein.

The isolation of the AT- and CT-containing purified leukotaxinepreparation by chromatography on hydroxyapatite as described in ExampleC1 is shown in the chromatogram of FIG. 4.

EXAMPLE C2 Chromatography of the AT- and CT-Containing Crude LeukotaxinePreparation B2 on Hydroxyapatite

Example C1 is repeated with the AT- and CT-containing crude leukotaxineprotein precipitate obtained as described in Example B2. According tothe description given in Example B2 after dialysis and removal of asmall fraction of insoluble proteins, 16 liters of clear leukotaxineprotein solution are obtained and applied to a column packed withhydroxyapatite with characteristics as given in Example C1 (volume 3×16liters, length-to-diameter ratio 10:1); i.e. with a diameter of 18 cmand a length of 180 cm. After elution, about 700 mg of the purifiedleukotaxine-containing product is obtained, which has an AT and CTportion of about 65% of total protein content.

EXAMPLE C3 Further Processing of the Purified Product C1 to an AT- andCT-Containing Highly Purified Leukotaxine Preparation byZone-Precipiration Chromatography

The AT- and CT-containing leukotaxine preparation obtained as given inExample C1 is dissolved in 0.1 mol/l sodium-potassium phosphate solutioncontaining 0.1 mol/l NaCl, 0.001 mol/l cysteine, 1 mol/l of ammoniumsulfate and having a pH of 7.4 to result a protein concentration ofabout 45 mg/ml. At a temperature of 4° C. the solution obtained (14 ml)is applied to a column packed with a molecular-sieve matrix of dextrancross-linked with epichlorohydrin (Sephadex G-50). The matrix isequilibrated with an ascending linear ammonium sulfate concentrationgradient of 1.0 to 3.2 mol/l (25 to 80% saturation) of ammonium sulfate.The gradient corresponds to a 2% increase in the ammonium sulfateconcentration per cm of column length. The gradient ranges over half ofthe column length. Thus, the other half of column length is equilibratedwith the buffer containing a constant concentration of 3.2 mol/lammonium sulfate. The length-to-diameter ratio of the column is 50:1,and the column volume has 100 fold volume of the solution applied. Theflow rate is 2 cm/h.

Protein elution from the column is done with the above-describedsodium-potassium phosphate buffer containing 1 mol/l of ammoniumsulfate. The active fractions are collected and the proteins areconcentrated in the usual manner (salting-out precipitation orultrafiltration).

For dissolving of the protein concentrate obtained, a buffer as given inexample C1 is preferably used (0.001 mol/l sodium-potassium phosphatebuffer containing 0.001 mol/l cysteine and having a pH of 7.2). 460 mg(10 ml) of purified AT- and CT-containing leukotaxine preparation isobtained, consisting of about 94% of AT and CT of total protein content.

EXAMPLE C4 Further Processing of Purified Product C1 to an AT- andCT-Containing, Highly Purified Leukotaxine Preparation by a Sequence ofRechromatography on Hydroxyapatite, Zone-Precipitation and AnalyticalMolecular Sieve Chromatography

The AT- and CT-containing, purified leukotaxine preparation obtained asgiven in Example C1 (650 mg; about 14 ml with 45 mg protein/ml) isdialysed against 0.001 mol/l sodiumpotassium phosphate buffer containing0.001 mol/l cysteine and having a pH of 7.2, ultrafiltrated, or desaltedby molecular sieve filtration (exclusion limit 1000 daltons). Then, asmall fraction os insoluble proteins is removed by centrifugation for 30min at 32,000×g and 4° C. The clear solution obtained isrechromatographed in the above buffer system on a column packed withhydroxyapatite which is equilibrated with the given buffer system. Thecolumn has a length-to-diameter ratio of 20:1 and at least a 5-foldvolume of protein solution applied. The elution is carried out with aphosphate concentration gradient as given in Example C1. Afterconcentration of active fractions pooled, 410 mg of highly purifiedleukotaxine preparation are obtained. About 91% of total protein contentare consisting of AT and CT.

This highly purified leukotaxine preparation obtained above is nowsubjected to a zone-precipitation chromatography as described in ExampleC3. After concentration of active fractions, 395 mg (10 ml) of furtherhighly purified AT- and CT-containing leukotaxine preparation areobtained. It now consists of about 96% of AT and CT.

Finally, this highly purified, AT- and CT-containing leukotaxinepreparation obtained as given by a sequence of chromatography andrechromatography on hydroxyapatite and zone-precipitation chromatographyis subjected to an analytical molecular sieve filtration. Theleukotaxine protein concentrate is dissolved in 0.003 mol/lsodium-potassium phosphate buffer containing 0.3 mol/l NaCl, 0.001 mol/lcysteine and having a pH of 7.4, to result an total maximum volume of 10ml with a protein concentration of about 35 to 40 mg/ml. The clearsolution obtained is applied to a column packed with Sephadex G-50having a particle size of 20 to 80 μm. The column used has at least a50-fold volume of the protein solution and a length-to-diameter ratio ofat least 50:1. For elution, a 0.03 mol/l sodium-potassium phosphatebuffer containing 0.3 mol/l NaCl, 0.001 mol/l cysteine, 0.4 mol/lammonium sulfate and having a pH of 7.4 is used (flow rate: 3 cm/h).After the usual concentration step for the active fractions, 370 mg (10ml) of a highly purified leukotaxine preparation are obtained. Itconsists of more than 99% of AT and CT (total yield: about 12%).

After desalting by dialysis, ultrafiltration, or molecular sievefiltration with an exclusion limit of 1000 daltons againstphysiological, buffered saline solution (e.g. 0.0015 mol/lsodium-potassium phosphate buffer containing 0.15 mol/l (0.9%) NaCl,0.001 mol/l cysteine and having a pH of 7.4), and after sterilization byfiltration (pore size 0.2 μm), the highly purified AT- and CT-containingleukotaxine preparation can be used for biological, physiological,pharmacological, and biochemical purposes.

EXAMPLE C5 Further Processing of the Purified AT- and CT-ContainingLeukotaxine Preparation C1 Into Separated, Individual AT and CT-Proteinsand to Their Molecular Homogeneity

The purified, AT- and CT-containing leukotaxine preparation obtained asdescribed in Example C1 (650 mg; about 14 ml with 45 mg protein/ml istransferred to a 0.003 mol/l sodium-potassium phosphate buffercontaining 0.3 mol/l NaCl, 0.001 mol/l cysteine and having a pH of 7.4,as given in Example C1 (final volume: 16 ml). After centrifugation at10,000×g, 4° C., for 30 min, a small quantity of insoluble materials isremoved. The clear solution obtained is applied to a column packed withSephadex G-50 with a particle size of 20 to 80 μm at a temperature of 4°C. The column has a 50-fold volume of the protein solution and alength-to-diameter ratio of 50:1. Elution is performed with a 0.03 mol/lsodium-potassium phosphate buffer containing 0.3 mol/l NaCl, 0.001 mol/lcysteine, 0.4 mol/l ammonium sulfate and having a pH of 7.4.

At a separation limit of 11,000 daltons, the eluate is introduced onto athreefold analytical molecular-sieve filtration cascade composed of twocolumns of the same specifications. Two main fractions with averagemolecular weights of 9500 daltons (130 mg AT) and 8500 daltons (325 mgCT) are obtained.

The two separated, individual protein fractions (AT and CT) obtained areconcentrated as usual by the addition of ammonium sulfate. Theprecipitated proteins are than dissolved separately in theabove-described buffer. The obtained clear solutions are individuallysubjected to analytical molecular sieve recycling chromatography underthe conditions given above for cascade chromatography.

The eluates are recycled three times at a separation limit of 10,000daltons (for AT) and 9000 daltons (for CT) as specified for cascadechromatography.

AT-recycling chromatography yields 113 mg of product with a molecularweight of 9500 daltons (AT) and 10 mg with a molecular weight of 8500daltons (CT). CT recycling chromatography delivers 312 mg of productwith a molecular weight of 8500 daltons (CT) and 15 mg with a molecularweight of 9500 daltons (AT). Each of the small residual amount of theproteins is combined with its main quantity. The two proteins are thenas usually individually concentrated (by ultrafiltration or salting-outprecipitation with ammonium sulfate) and the protein precipitatesdissolved as described in Example C1.

Then, the two protein fractions (AT and CT) are subjected separately toa final purification consisting of three steps as described in ExampleC4. The same conditions are used as for the joint purification of AT andCT within the leukotaxine preparation. The sequence of the three stepsconsists of one rechromatography on hydroxyapatite, onezone-precipitation chromatography, and one analytical molecular sievechromatography for each of the two proteins (AT and CT).

For anaphylatoxin, 100 mg of the AT-protein are obtained (about 10%total yield). The preparation consists of more than 99% of AT. Proteincontaminations cannot be detected on the basis of electrophoretic,chromatographic, immunoanalytical and solubility criteria. The ATpreparation can thus be considered as molecularly homogeneous.

For cocytotaxine, 250 mg of the CT-protein are obtained (about 11% totalyield). The preparation consists of more than 99% of cocytotaxine.Protein contaminations cannot be detected on the basis ofelectrophoretic, chromatographic, immunoanalytical and solubilitycriteria. The CT preparation can as well be considered as molecularlyhomogeneous.

The separation of the AT- and CT-containing leukotaxine preparation intothe proteins AT and CT and their further individual purification tomolecular homogeneity as described in Example C5 are shown in thechromatograms presented in FIGS. 5 to 13.

As already mentioned, the absolute properties and quantities yielded andthe ratio of the proteins AT and CT obtained in the process may varysomewhat with the origin and contact-activation method of blood and theanimal species used.

Thus, for example, as is known classical anaphylatoxin derived fromnormal human serum has only a very low spasmogenic activity on smoothmuscles when compared to classical AT derived from normal porcine, rator guinea pig serum. However, the combination of AT and CT has a highchemotactic activity for neutrophil leucocytes which is comparable tothe protein combination derived from normal porcine, rat and guinea pigblood. The processes for obtaining AT and CT, however, are notrestricted for use with normal, contact-activated mammalian serum. Ifprotease inhibitors (like 6-amino caproic acid,di-isopropyl-fluoro-phosphate, etc.) are added for special purposes tothe sera, process examples B2, B3, B5, B8 are especially suitable for aninitial, crude removal of contaminating foreign proteins, followed by asuitable selection of process examples C for obtaining highly purifiedproteins. Due to the known action of some of such protease inhibitors onserum encyme systems, and depending on conditions of contact-activationof the sera, modified, natural protein analogues of AT and CT formed asintermediate in sequences of steps of the AT- and CT-formation processcan as well be isolated as fractions in the process of the invention.

After transfer of the two individual proteins AT and CT into aphysiological buffered saline solution and after their sterilization asdescribed in Example C4, the two individual proteins can be used forappropriate biochemical, physiological, pharmacological, or biologicalpurposes. Or, each can be crystallized according to the known methods(J. H. Wissler, op. cit.).

What is claimed is:
 1. In a process for producing and obtaining ananaphylatoxin- and cocytotaxin-containing leucotaxine preparation inbiologically active form from contact-activated mammalian serum, theprocess including separating the proteins from other serum constituentsto obtain a serum protein concentrate fraction, separating a part ofaccompanying foreign blood proteins from anaphylatoxin and cocytotaxinpresent in said protetin concentrate fraction, and isolating theleocotaxine preparation by chromatography on hydroxyapatite, wherein theimprovement comprises separating, prior to chromatography onhydroxyapatite, a major fraction of accompanying foreign bloodconstituents from said serum protein concentrate by fractional elutionor precipitation with a watersoluble alcohol and at least one molecularsieve filtration.
 2. In a process for producing and obtaininganaphylatoxin and cocytotaxin proteins in molecularly homogeneous,biologically active form from contact-activated mammalian serum, theprocess comprising separating the proteins from other serum constituentsto obtain a serum protein concentrate fraction, separating a part ofaccompanying foreign blood proteins from anaphylatoxin and cocytotaxinpresent in said protein concentrate fraction, isolating the leucotaxinepreparation by chromotography on hydroxyapatite, and separating theleucotaxine preparation into anaphylatoxin and cocytoxin proteins bychromatographic methods, the improvement comprising separating, prior tochrmoatography on hydroxyapatite, a major fraction of accompanyingforeign blood constituents from said serum protein concentrate fractionby fractional elution, and precipitation with a water-soluble alcoholand at least one molecular sieve filtration.
 3. In a process forproducing and obtaining an anaphylatoxin- and cocytotaxin-containingleucotaxine preparation in biologically active form fromcontact-activated mammalian serum, the process including separatingproteins from other serum constituents to obtain a serum proteinconcentrate fraction, separating a part of accompanying foreign bloodproteins from anaphylatoxin and cocytotaxin present in said proteinconcentrate fraction, and isolating the luecotaxine preparation bychromotography on hydroxyapatite, wherein the improvement comprisesseparating prior to chromatography on hydroxyapatite, a major fractionof accompanying foreign blood constitutents from said serum proetinconcentrate fraction after dissolving it in a protein-compatible liquidby a protein precipitation step with a water-soluble alcohol and thenfiltering the supernatant protein solution containing anaphylatoxin andcocytotaxin by molecular sieve filtration.
 4. A process according toclaim 3, wherein the supernatant protein solution obtained afterprecipitation of foreign proteins with a water-soluble alcohol is thensubjected at least once to preparative molecular sieve filtration andonce to analytical molecular sieve filtration in water or in aprotein-compatible liquid.
 5. A process according to claim 3, whereinsaid protein-compatible liquid is 0.001 mol/l sodium potassium phosphatebuffer containing about 0.1 mol/l NaCl and having pH of approximately6.3.
 6. A process according to claims 3 or 4, wherein a firstpreparative molecular sieve filtration is carried out at a temperatureof 0° to 8° C. and a pH of 6 to 8 on a hydrophilic, water-swellingmolecular sieve having a separation limit of 20,000 daltons.
 7. Aprocess according to claim 3 or 4, wherein a second preparative moleculesieve filtration is carried out at a temperature of 0° to 8° C. and a pHof 6 to 8 on a hydrophilic, water-swelling molecular sieve havingseparation limit of 13,000 daltons.
 8. A process according to claim 4,wherein the analytical molecular sieve filtration is carried out at atemperature of 0° to 8° C. and a pH of 6 to 8 on a hydrophilicwater-swelling molecular sieve with separation limits of 11,000 and6,000 daltons.
 9. A process according to claim 6, wherein the molecularsieve filtration is carried out in the presence of ammonium sulfate at aconcentration of up to about 0.6 mol/l.
 10. A process according to claim7, wherein the molecular sieve filtration is carried out in the presenceof ammonium sulfate at a concentration of up to about 0.6 mol/l.
 11. Aprocess according to claim 8, wherein the molecular sieve filtration iscarried out in the presence of ammonium sulfate at a concentration of upto about 0.6 mol/l.
 12. A process according to claim 6, wherein themolecular sieve filtration is carried out in the presence of cysteine ata concentration of up to about 0.001 mol/l.
 13. A process according toclaim 7, wherein the molecular sieve filtration is carried out in thepresence of cysteine at a concentration of up to about 0.001 mol/l. 14.A process according to claim 8, wherein the molecular sieve filtrationis carried out in the presence of cysteine at a concentration of up toabout 0.001 mol/l.
 15. A process according to claim 3, wherein ethanolis used as the water-soluble alcohol.
 16. In a process for producing andobtaining an anaphylatoxin- and cocytotaxin-containing leucotaxinepreparation in biologically active form from contact-activated mammalianserum, the process including separating proteins from other serumconstituents to obtain a serum protein concentrate fraction, separatinga part of accompanying foreign blood proteins from anaphylatoxin andcocytotaxin present in said protein concentrate fraction, and isolatingthe leucotaxine preparation by chromatography on hydroxyapatite, whereinthe improvement comprises separating prior to chromatography onhydroxyapatite, a major fraction of accompanying foreign bloodconstituents from said serum protein concentrate fraction by afractional elution step which is followed, after dissolving the residualprotein precipitate in a protein-compatible liquid, by a proteinprecipitation step with a water soluble alcohol and then filtering thesupernatant protein solution containing anaphylatoxin and cocytotaxin bymolecular sieve filtration.
 17. A process according to claim 16, whereinthe supernatant protein solution obtained after precipitation of foreignproteins with a water-soluble alcohol is then subjected twice topreparative molecular sieve filtration and once to analytical molecularsieve filtration in water or in a protein-compatible liquid.
 18. Aprocess according to claims 16 or 17 wherein for fractional elution, theserum protein concentrate fraction is adjusted to an ammonium sulfateconcentration of approximately 2.6 mol/l with a protein-compatibleliquid.
 19. A process according to claim 18, wherein saidprotein-compatible liquid is 0.001 mol/l sodium potassium phosphatebuffer containing about 0.1 mol/l NaCl and having a pH of approximately6.3.
 20. A process according to claims 16 or 17, wherein a firstpreparative molecular sieve filtration is carried out at a temperatureof 0° to 8° C. and a pH of 6 to 8 on a hydrophilic, water-swellingmolecular sieve having a separation limit of 20,000 daltons.
 21. Aprocess according to claims 16 or 17, wherein a second preparativemolecular sieve filtration is carried out at a temperature of 0° to 8°C. and a pH of 6 to 8 on a hydrophilic, water-swelling molecular sievehaving separation limit of 13,000 daltons.
 22. A process according toclaim 17, wherein the analytical molecular sieve filtration is carriedout at a temperature of 0° to 8° C. and a pH of 6 to 8 on a hydrophilicwater-swelling molecular sieve with separation limits of 11,000 and6,000 daltons.
 23. A process according to claim 20, wherein themolecular sieve filtration is carried out in the presence of ammoniumsulfate at a concentration of up to about 0.6 mol/l.
 24. A processaccording to claim 21, wherein the molecular sieve filtration is carriedout in the presence of ammonium sulfate at a concentration of up toabout 0.6 mol/l.
 25. A process according to claim 22, wherein themolecular sieve filtration is carried out in the presence of ammoniumsulfate at a concentration of up to about 0.6 mol/l.
 26. A processaccording to claim 20, wherein the molecular sieve filtration is carriedout in the presence of cysteine at a concentration of up to about 0.001mol/l.
 27. A process according to claim 21, wherein the molecular sievefiltration is carried out in the presence of cysteine at a concentrationof up to about 0.001 mol/l.
 28. A process according to claim 22, whereinthe molecular sieve filtration is carried out in the presence ofcysteine at a concentration of up to about 0.001mol/l.
 29. A processaccording to claim 16, wherein ethanol is used as the water-solublealcohol.
 30. In a process for producing and obtaining anaphylatoxin andcocytoxain proteins in molecularly homogeneous, biologically active formfrom contact-activated mammalian serum, the process comprisingseparating the proteins from other serum constituents to obtain a serumprotein concentrate fraction, separating a part of accompanying foreignblood proteins from anaphylatoxin and cocytotaxin present in the saidprotein concentrate fraction, isolating the leucotaxine preparation bychromatography on hydroxyapatite, and separating the leucotaxinpreparation into anaphylatoxin and cocytotaxin proteins bychromatographic methods, the improvement comprising separating, prior tochromatography on hydroxyapatite, a major fraction of accompanyingforeign blood constitutents from said serum protein concentrate fractionafter dissolving it in a protein-compatible liquid by a proteinprecipitation step with a water-soluble alcohol and then filtering thesupernatant protein solution containing anaphylatoxin and cocytotaxin bymolecular sieve filtration.
 31. A process according to claim 30, whereinthe supernatant protein solution obtained after precipitation of foreignproteins with a water-soluble alcohol is then subjected at least once topreparative molecular sieve filtration and once to analytical molecularsieve filtration in water of in a protein-compatible liquid.
 32. Aprocess according to claim 30, wherein the protein-compatible liquid isa 0.001 mol/l sodium potassium phosphate buffer containing about 0.1mol/l NaCl and having a pH of approximately 6.3.
 33. A process accordingto claim 31 or 32, wherein a first preparative molecular sievefiltration is carried out at a temperature of 0° to 8° C. and a pH of 6to 8 on a hydrophilic, water-swelling molecular sieve having aseparation limit of 20,000 daltons.
 34. A process according to claim 31or 32, wherein second preparative molecular sieve filtration is carriedout at a temperature of 0° to 8° C. and a pH of 6 to 8 on a hydrophilic,water-swelling molecular sieve having a separation limit of 13,000daltons.
 35. A process according to claim 32, wherein the analyticalmolecular sieve filtration is carried out at a temperature of 0° to 8°C. and a pH of 6 to 8 on a hydrophilic water-swelling molecular sievewith separation limits of 11,000 and 6,000 daltons.
 36. A processaccording to claim 33, wherein the molecular sieve filtration is carriedout in the presence of ammonium sulfate at a concentration of up toabout 0.6 mol/l.
 37. A process according to claim 34, wherein themolecular sieve filtration is carried out in the presence of ammoniumsulfate at a concentration of up to about 0.6 mol/l.
 38. A processaccording to claim 35, wherein the molecular sieve filtration is carriedout in the presence of ammonium sulfate at a concentration of up toabout 0.6 mol/l.
 39. A process according to claim 33, wherein themolecular sieve filtration is carried out in the presence of cysteine ata concentration of up to about 0.001 mol/l.
 40. A process according toclaim 34, wherein the molecular sieve filtration is carried out in thepresence of cysteine at a concentration of up to about 0.001 mol/l. 41.A process according to claim 35, wherein the molecular sieve filtrationis carried out in the presence of cysteine at a concentration of up toabout 0.001 mol/l.
 42. A process according to claim 31, wherein ethanolis used as the water-soluble alcohol.
 43. The process of claim 31,wherein said leucotaxine preparation is separated into anaphylatoxin andcocytotaxin using cascade or recyling molecular sieve filtration.
 44. Aprocess for obtaining an anaphylatoxin and cocytotaxin containingleucotaxin preparation in biologically active form from contactactivated mammalian serum, comprising:(a) separating serum proteins fromother serum constituents to obtain a first protein concentrate; (b)separating a major fraction of foreign blood proteins from said serumprotein concentrate by dissolving the protein concentrate in aprotein-compatible liquid, carrying out a protein precipitation stepwith a water-soluble alcohol to obtain a protein solution containinganaphylatoxin and cocytotaxin; (c) filtering said protein solution withmolecular sieves to obtain a second protein concentrate; and (d)isolating said anaphylatoxin and cocytotaxin containing preparation fromsaid second protein concentratrate using hydroxyapatite chromatography.45. The invention of claim 44 wherein said anaphylatoxin and cocytotaxincontaining leucotaxin preparation is separated into anaphylatoxin andcocytotaxin proteins.
 46. A process for obtaining anaphylatoxin andcocytotaxin proteins in molecularly homogeneous, biologically activeform from contact activated mammalian serum, comprising:(a) separatingserum proteins from other serum constituents to obtain a first proteinconcentrate; (b) separating a major fraction of foreing blood proteinsfrom said serum protein concentrate by dissolving the proteinconcentrate in a protein-compatible liquid, carrying out a proteinprecipitation step with a water-soluble alchol to obtain a proteinsolution containing anaphylatoxin and cocytotaxin; (c) filtering saidprotein precipitate with molecular sieves to obtain a second proteinconcentrate; (d) isolating said anaphylatoxin and cocytotaxin containingpreparation from said second protein concentrate using hydroxyapatitechromatography, and (e) further purifying said preparation to obtainanaphylatoxin and cocytotaxin proteins.
 47. In a process for producingand obtaining anaphylatoxin and cocytotaxin proteins in molecularlyhomogeneous, biologically active form from contact-activated mammalianserum, the process comprising separating the proteins from other serumconstituents to obtain a serum protein concentrate fraction, separatinga part of accompanying foreign blood proteins from anaphylatoxin andcocytotaxin present in the said protein concentrate fraction, isolatingthe leucotaxine preparation by chromatography on hydroxyapatite, andseparating the leucotaxine preparation into anaphylatoxin andcocytotaxin proteins by chromatographic methods the improvementcomprising separating, prior to chromatography on hydroxyapatite, amajor fraction of accompanying foreign blood constituents from saidserum protein concentrate fraction by a fractional elution step whichfollowed, after dissolving the residual protein precipitate in aprotein-compatible liquid, by a protein precipitating step with awater-soluble alcohol and then filtering the spernatant protein solutioncontaining anaphylatoxin and cocytotaxin by molecule sieve filtration.48. A process according to claim 47, wherein the supernatant proteinsolution obtained after precipitation of foreign proteins with awater-soluble alcohol is then subjected twice to preparative molecularsieve filtration and once to analytical molecular sieve filtration inwater of in a protein-compatible liquid.
 49. A process according toclaims 47 or 48, wherein for fractional elution, the serum proteinconcentrate fraction is adjusted to an ammonium sulfate concentration ofapproximately 2.6 mol/l with a protein-compatible liquid.
 50. A processaccording to claim 49, wherein the protein-compatible liquid is a 0.001mol/l sodium potassium phosphate buffer containing about 0.1 mol/l NaCland having a pH of approximately 6.3.
 51. A process according to claim47 or 48, wherein a first preparative molecular sieve filtration iscarried out at a temperature of 0° to 8° C. and a pH of 6 to 8 on ahydrophilic, water-swelling molecular sieve having a separation limit of20,000 daltons.
 52. A process according to claim 47 or 48, wherein asecond preparative molecular sieve filtration is carried out at atemperature of 0° to 8° C. and a pH of 6 to 8 on a hydrophilic,water-swelling molecular sieve having a separation limit of 13,000daltons.
 53. A process according to claim 48, wherein the analyticalmolecular sieve filtration is carried out at a temperature of 0° to 8°C. and a pH of 6 to 8 on a hydrophilic water-swelling molecular sievewith separation limits of 11,000 and 6,000 daltons.
 54. A processaccording to claim 51, wherein the molecular sieve filtration is carriedout in the presence of ammonium sulfate at a concentration of up toabout 0.6 mol/l.
 55. A process according to claim 52, wherein themolecular sieve filtration is carried out in the presence of ammoniumsulfate at a concentration of up to about 0.6 mol/l.
 56. A processaccording to claim 53, wherein the molecular sieve filtration is carriedout in the presence of ammonium sulfate at a concentration of up toabout 0.6 mol/l.
 57. A process according to claim 51, wherein themolecular sieve filtration is carried out in the presence of cysteine ata concentration of up to about 0.001 mol/l.
 58. A process according toclaim 52, wherein the molecular sieve filtration is carried out in thepresence of cysteine at a concentration of up to about 0.001 mol/l. 59.A process according to claim 53, wherein the molecular sieve filtrationis carried out in the presence of cysteine at a concentration of up toabout 0.001 mol/l.
 60. A process according to claim 47, wherein ethanolis used as the water-soluble alcohol.
 61. The process of claim 47,wherein said leucotaxine preparation is separated into anaphylatoxin andcocytotaxin using cascade or recycling molecular sieve filtration.
 62. Aprocessfor obtaining an anaphylatoxin and cocytotaxin containingleucotaxine preparation in biologically active form from contactactivated mammalian serum, comprising:(a) separating serum proteins fromother serum constituents to obtain a first protein concentrate; (b)separating a major fraction of foreign blood proteins from said serumprotein concentrate by fractional elution to obtain a proteinprecipitate containing anaphylatoxin and cocytotaxin, dissolving saidprotein prepitate in a protein-compatible liquid, carrying out a proteinprecipitation step with a water-soluble alcohol to obtain a proteinsolution containing anaphylatoxin and cocytotaxin; (c) filtering saidprotein solution with molecular sieves to obtain a second proteinconcentrate; (d) isolating said anaphylatoxin and cocytotaxin containingpreparation from said second protein concentrate using hydroxyapatitechromatography.
 63. The invention of claim 62 wherein said anaphylatoxinand cocytotaxin containing leucotaxin preparation is separated intoanaphylatoxin and cocytotaxin proteins.
 64. A process for obtaininganaphylatoxin and cocytotaxin proteins in molecularly homogenous,biologically active form from contact activated mammalian serum,comprising:(a) separating serum proteins from other serum constituentsto obtain a first protein concentrate; (b) separating a major fractionof foreign blood proteins from said serum protein concentrate byfractional elution to obtain a protein precipitate containinganaphylatoxin and cocytotaxin, dissolving said protein precipitate in aprotein-compatible liquid, carrying out a protein precipitation stepwith a water-soluble alcohol to obtain a protein solution containinganaphylatoxin and cocytotaxin; (c) filtering said protein precipitatewith molecular sives to obtain a second protein concentrate; (d)isolating said anaphylatoxin and cocytotaxin containing preparation fromsaid second protein concentrate using hydroxyapatite chromatography, and(e) further purifying said preparation to obtain anaphylatoxin andcocytotaxin proteins.